This project will examine a novel method of delivery of proteins into mammalian cells, and it specifically targets the area of investigation related to the HIV envelope protein gp120. Eliciting a strong CD4 T cell stimulation and cytotoxic T lymphocyte (CTL) response against HIV-1 and HIV-1 infected cells should be a major objective of any HIV vaccine development work. One way to achieve this objective is to develop a more efficient but safe antigen delivery system. The current approaches to deliver HIV immunogens using live vector systems are not adequate. We have developed a peptide-based delivery system that may overcome the limitations of existing delivery vectors. This project will serve as a proof-of-the-concept project using gp120 to study the efficiency of protein delivery, intracellular processing, and antigen presentation mediated by a 22-amino acid peptide derived from a surface protein of Mycobacterium tuberculosis. The peptide called Inv3 was derived from a sequence based on the most active domain of a protein called mycobacterium cell entry protein or Mcep. Mcep and Inv3 are able to deliver materials (microspheres, colloidal gold particles) complexed to them into nonphagocytic cells with very high efficiency. We have constructed an expression plasmid (pInv3) that facilitates expression of any protein with Inv3 peptide fused to the N-terminus. We have already shown that beta-galactosidase, green fluorescent protein, and ovalbumin expressed in fusion with Inv3 are able to abundantly associate with HeLa cells. We plan to express gp120 using pInv3 to deliver the protein into mammalian cells to study the efficiency of its delivery and the intracellular location of the protein. The two specific aims are as follows: 1) study the efficiency of cell delivery of Inv3-gp120 fusion protein and its final destination inside nonphagocytic cells and 2) evaluate the presentation of peptides derived from a known protein (ovalbumin) expressed in fusion with Inv3 by antigen presenting cells (APCs) via MHC class I and class II pathways, using APCs that have been specifically designed to express a) Kb allele in HeLa cells for MHC class I antigen presentation, and b) Ak specific L cells for MHC class II antigen presentation. The intracellular delivery, processing, and antigen presentation facilitated by Inv3-ovalbumin will be compared to those associated with a vaccinia vector delivered ovalbumin. If Inv3 is demonstrated to enhance the delivery of gp120 and facilitate ovalbumin peptide presentation by APCs superior to that mediated by the vaccinia vector system, we will enter a full collaboration with the HIVNET program at the California Department of Health Services to apply this technology to human cells as part of our long-term goal, for which we will seek additional funding support. The successful outcome of this project may not only identify potential vaccine candidates for HIV-1, but may serve as a proof-of-concept study for a completely novel approach to vaccine development. This ability to deliver any polypeptide into cells enables one to screen the entire coding capacity of a genome of a pathogen, without any preconceived bias, for immunodominant or immunoprotective peptide sequences. The ability to identify such domains may open a completely new direction in identifying peptides that may be able to elicit protective CTL response against not just intracellular pathogens, but against tumor cells. While this may be a high risk project, the yield, if shown to work, even as a method to enhance mammalian cell delivery of proteins, should have a significant impact.